Vaccination against bacterial infections is an important medical pursuit, representing a preventive medical intervention recommended for virtually every individual. Design of vaccines to combat bacterial infection or the pathogenesis of bacterial infection often targets bacterial proteins, such as toxin produced by a bacterium. Such is the case, for example, in vaccines against anthrax, diphtheria, and tetanus. Another vaccine approach targets the outer capsule of a bacterium, however many of the antigens comprising a bacterial pathogen's capsule layer stimulate little or no long-term immune response, which complicates their use in creating effective vaccines. Capsules make up the outer surface of many bacteria and are typically composed of polymers of organic compounds such as carbohydrates, amino acids, or alcohols. Capsules are quite diverse chemically. For polysaccharide-based capsules the sugar units can be linked together in various molecular configurations and can be further substituted with phosphate, nitrogen, sulfate, and other chemical modifications. Capsules may be a virulence factor, by inhibiting microbes from being efficiently phagocytosed and killed by host macrophages and polymorphonuclear leukocytes.
Antibodies against capsules provide a potent defense against encapsulated organisms by mediating complement fixation on the microbial surface, which can result in bacterial lysis or opsonization, uptake, and killing by phagocytic host immune cells. The most potent antibodies against microbial capsules are IgG antibodies. Capsular antigens are generally classified as T-independent antigens as they elicit immune responses that do not involve T-cell help and therefore do not elicit long-lasting immunological memory responses. However, the covalent coupling of a protein to a capsular antigen renders the capsular antigen “T-dependent”, and such T-dependent antigens then elicit a helper T cell-mediated (Th-dependent) IgG-based memory B-cell, or anamnestic, response.
Various methods for rendering vaccine antigens more immunogenic and ideally T-dependent have been studied. Most bacterial surface polysaccharides are immunogenic by themselves and are capable of eliciting an immune response that will recognize the naturally occurring antigen in the microbial capsule. However, when the capsular polysaccharides alone are used as vaccines, they generally do not promote long-lasting immunity, nor are they very effective in immunizing children under the age of 2. It has been demonstrated that covalently linking a polysaccharide antigen to a carrier protein can greatly increase immunogenicity of the polysaccharide and promote the desired T-dependent immune response (or immune memory) that leads to protection of the host against subsequent infections by the antigen-bearing microorganism. For example, an unconjugated pneumococcal vaccine, such as Merck's Pneumovax®, is efficacious against invasive pneumococcal disease in individuals, however it is ineffective (e.g., in infants) at eliciting immunological memory and the desired protective immunity that would elicit long-term immunity and avoid the necessity of repeated immunizations. Conjugate pneumococcal vaccines such as Pfizer's Prevnar® (Pfizer Inc., USA), have been shown to be highly immunogenic even in 2-month old infants, induce T-dependent immunity and to be highly efficacious.
However, while conjugate vaccines are promising immunologically, they can be extremely difficult and complicated (and expensive) to manufacture, greatly deterring their distribution to those in need of vaccination throughout the world. For example, in the case of Prevnar®7, each S. pneumoniae strain used to provide the seven polysaccharide antigens used for conjugation is grown in a bioreactor; the cells are harvested; polysaccharide is extracted, purified, hydrolyzed to the appropriate size; the individual antigens are then conjugated to a protein carrier; the conjugate is re-purified, mixed with the additional 6 other polysaccharide-protein complexes (conjugates) that were prepared in a similar manner; and the multi-conjugate mixture is finally adjuvanted with alum. It is estimated that there are more than 200 GMP steps in the manufacture of the heptavalent Prevnar® vaccine.
Recently, protein matrix vaccines have been proposed as an alternative to conjugate vaccines. See, US published application no. US-2008-0095803 (Mekalanos, J.), published Apr. 24, 2008; international patent application publication no. WO 2008/021076 (Mekalanos, J.), published Feb. 21, 2008; and international patent application publication no. WO 2011/031893 (Killeen, K., et. al.), published Mar. 17, 2011), incorporated herein by reference. Rather than covalently conjugating an antigen of interest to a carrier, a protein matrix vaccine entraps the antigen in a carrier protein matrix, prepared by cross-linking the carrier protein in the presence of the desired antigen. Significant covalent linking of the antigen to the carrier protein is avoided; rather, the antigen remains associated with the matrix by becoming entrapped by the protein carrier during matrix formation (cross-linking reaction). Such protein matrix vaccines have been demonstrated to elicit greater immunogenicity than vaccines prepared using the antigen alone; and protein matrix vaccines may also elicit the sort of immune response (i.e., induction of T-dependent immunity) seen with conjugate vaccines. Synthesis of protein matrix vaccines does not involve complicated conjugation reactions, and typically requires fewer processing steps, which makes the protein matrix vaccines, in turn, less expensive to manufacture than a conjugate vaccine.
Although protein matrix vaccines provide several advantages, the titer of antigen-specific antibodies elicited by protein matrix vaccines is often lower than the titer elicited by a corresponding conjugate vaccine. WO 2011/031893 teaches that separating the protein matrix vaccines by size exclusion chromatography and selecting the fractions containing high molecular weight protein matrix particles for immunization can lead to titers similar to those elicited by conjugated polysaccharide vaccines. However, it is a persistent technical problem in the field to provide a means for producing protein matrix vaccines of increased immunogenicity, in order to exploit the scientific promise and the manufacturing and cost advantages of this emerging technology. There is a continuing need for improved protein matrix vaccines having enhanced immunogenicity or potency.